Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with Kaposi's sarcoma (KS) and several other AIDS-related malignancies. The long-term goal of this project is to apply effective KSHV genetic and infection systems to delineate the molecular mechanism of KSHV-induced pathogenesis. In previous funding period, we have developed and refined a KSHV genetic manipulation system and a KSHV infection model of primary human umbilical vein endothelial cell (HUVEC). We have used these model systems to successfully define the functions of KSHV genes and examine KSHV-cell interactions. These studies have shown that both KSHV latent and lytic replication phases are important for KSHV-induced malignant transformation. Thus, defining the mechanism controlling KSHV latency and lytic replication is a critical step in delineating the pathogenesis of KSHV-induced malignancies. Toward this goal, our recent studies have shown that KSHV-encoded microRNAs (miRs) promote viral latency by inhibiting viral lytic replication program. To further understand the functions of KSHV miRs, we have developed novel computational algorithms for miR target prediction and time-series data analysis for complex signaling networks. The objective of this renewal application is to continue to define the functions of KSHV miRs in viral infection and replication, and delineate the underlying molecular mechanisms. Our hypothesis is that KSHV miRs regulate viral lifecycle by targeting viral and/or cellular genes to inhibit specific stages of KSHV infection and replication. To test this hypothesis and accomplish the objective, we will carry out the following three specific aims: (1) To define the stages of viral infection and replication regulated by KSHV miRs by examining virus production, viral gene expression, viral DNA replication, and virion packaging and egress; (2) To identify the specific viral miRs that regulate KSHV infection and replication by genetic complementation and loss-of-function approaches using miR suppressors or genetic mutants; and (3) To delineate the mechanisms by which KSHV miRs regulate viral infection and replication by identifying the direct viral and cellular targets using a systems biology approach. The proposed project is significant because it will, for the first time, define the roles of KSHV miRs in viral infection and replication, and delineate the underlying mechanisms. The proposed study is multidisciplinary in nature, and innovative through the integration of novel computational, genetic, molecular, cellular, microarray and proteomic approaches. The successful completion of this project will lead to the establishment of a novel systems biology approach for delineating the functions of viral and cellular miRs using KSHV as a model system. The outcomes will not only provide insights into the mechanism of KSHV latency and replication but also shed light on the pathogenesis of KSHV-induced malignancies. PUBLIC HEALTH RELEVANCE: Kaposi's sarcoma is a common malignancy in AIDS patients in US and worldwide inflicting morbidity and mortality to the society. This project will investigate the mechanism underlining the development of Kaposi's sarcoma, and identify potential targets for the prevention and treatment of this disease.